Although breast cancer is one of the most common malignancies in American women, the genetic mechanisms responsible for its pathogenesis and clinical course remain largely unclear. TBX3 is a T-box transcription factor that may play a role in breast cancer and has not been previously investigated. Mutations of TBX3 in humans and mice show the clinical features of hypoplastic or absence of the mammary gland. Our earlier studies demonstrate that TBX3 is overexpressed in breast cancer cell lines, and can immortalize and transform mouse embryo fibroblast cells. TBX3 can also inhibit the expression of the p14ARF gene, a tumor suppressor and an inhibitor of MDM2-mediated degradation of p53. Therefore, overexpression of TBX3 decreases the stability of p53. Most recently, TBX3 was found to be elevated in serum of the patients with breast cancer. Collectively, these findings suggest that TBX3 plays an important role in breast cancer. In this proposal, we show that TBX3 interacts with histone deacetylase (HDAC)1, 2 and 5. Our central hypothesis is that TBX3 overexpression is associated with breast cancer and that TBX3 recruits HDACs to the p14ARF promoter and inhibits p14ARF tumor suppressor expression causing breast cancer. Aim 1, we will examine whether TBX3 is overexpressed in primary breast cancer tissues. Expression levels of TBX3 will be correlated with clinical outcomes and other established biomarkers. We anticipate that TBX3 could be a novel biomarker for breast cancer. TBX3 is mechanistically implicated in tumor growth, presumably by inhibiting the expression of p14ARF tumor suppressor gene. Although TBX3 has been characterized as a transcriptional repressor, the mechanism by which TBX3 represses gene expression is still unknown. Our preliminary results show that TBX3 interacts with HDACs and that the interaction is physiologically important. For Aim 2, we hypothesize that TBX3 recruits HDACs to p14ARF promoter and represses p14ARF tumor suppressor gene expression. We will test the interaction between TBX3 and HDACs by a series of in vivo and in vitro assays, including immunohistochemical analysis, Glutathione-S-transferase (GST) pulldown assay and chromatin immunoprecipitation (CHIP). Physiological significance of the TBX3-HDAC interaction will be further investigated in breast cancer cells and with the p14ARF promoter. In Aim 3, we propose to identify the TBX3 direct targets via chromatin immunoprecipitation-guided ligation selection (CHIP-GLAS). The CHIP-GLAS assay combines chromatin precipitation and microarray, which could be used to identify 20,000 promoter DNA/transcription factor interactions in one experiment with high sensitivity and specificity. Using this approach, we found that TBX3 binds to more than 600 promoters. We will verify those target genes involved in breast cancer and mammary gland development using various in vivo and in vitro assays. An important feature of the proposed research is translational for the development of a novel biomarker for breast cancer and basic biological information to clinical application will be optimized. Elucidation of the TBX3- HDAC interaction will deepen our understanding of the functions of TBX3 and may also lead to the identification of a novel therapeutic target for breast cancer.